Method and device for transporting or binding-specific separation of electrically charged molecules

ABSTRACT

Electrically charged molecules need to be transported in order to create a DNA sensor. The following measures are undertaken: base metals are introduced into a solution as a positive ion; negatively charged molecules are transported in an opposite direction and are enriched in the vicinity of the measuring electrodes. Binding-specific separation of the charged molecules can be achieved by forming metal layers on the measuring electrodes by depositing metal ions from the solution when a suitable potential is selected. Target DNA can more particularly be introduced into the vicinity of the catcher molecules on the measuring electrodes and non-specifically bound DNA can be removed. According to the associated device, the electrode arrangement may be associated with a sacrificial electrode made of more base metal than the material of the measuring electrodes. The measuring electrodes in particular may be made of noble metal, preferably gold, and the sacrificial electrode may be made of cooper.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/DE2003/003938 which has anInternational filing date of Nov. 28, 2003, which designated the UnitedStates of America and which claims priority on German Patent Applicationnumber DE 102 56 415.9 filed Dec. 2, 2002, the entire contents of whichare hereby incorporated herein by reference.

FIELD OF THE INVENTION

The invention generally relates to a method for transporting orbinding-specific separation of electrically charged molecules in anaqueous solution, in particular during the operation of a DNA sensorwith a redox cycling process between measuring electrodes. The inventionadditionally generally relates to the associated devices.

BACKGROUND

The transporting of charged particles in an electric field (migration)plays an important part in numerous methods of molecular biology. Themigration velocity v of the charged particles in the liquid medium is inthis case proportional to the field strength E and the ion charge Q andinversely proportional to the particle radius r and the viscosity η ofthe suspension. The following results for the velocity v:v=QE/6πrη  (1)

During electrophoresis, by way of example, biomolecules, i.e primarilyproteins and DNA, which differ with regard to their size and/or chargeare separated from one another. The presence of other mobile chargedparticles is to be avoided in certain forms of electrophoreticseparation (e.g. isoelectric focusing) since otherwise the chargetransport is undertaken partly or wholly by these particles and not bythe molecules to be separated. Therefore, amino acids that have theirisoelectric point at the desired pH value are often used as a buffer.That is to say that, at the pH value set, the buffer moleculesthemselves have no net charge and are therefore not subject tomigration.

Electric fields are also used in the transporting of charged molecules,e.g. in order to increase or to decrease the concentration at a specificlocation. Particularly in the case of microsensors, e.g. for DNAanalysis, it is possible to increase the sensitivity if the DNAfragments (target molecules) to be detected are concentrated at thelocation of the capture molecules (sensor surface). The number ofcapture/target molecule bonds thus increases in accordance with the lawof mass action. In any event, however, during such a reaction not onlyare capture/target molecule pairs formed which match one another exactlybut also those whose sequence do not correspond to one another exactlyat some sites (mismatches).

Since the magnitude of the binding energy decreases with the number ofnon-corresponding bases, those bonds which have a specific number ofmismatches can be separated again selectively by the application ofappropriate forces (stringency treatment). As force, it is possible herefor an electric field to take effect which has an opposite polarity incontrast to the first process, the concentration of the molecules.

A prerequisite for transporting charged particles in the electric fieldis a field gradient that has a strictly monotonic profile within theelectrolyte or the transport path. That is to say that the fieldgradient must not change its sign and must not become zero. Theapplication of an arbitrary voltage is not necessarily sufficient forthis purpose for aqueous systems.

In the absence of a chemical reaction before the electrodes, the voltagedrops across the electrochemical double layer and the field gradientbetween the electrodes becomes zero. However, if a reduction oroxidation reaction takes place at the electrodes, the double layerbefore the electrodes is depolarized and the electric field has astrictly monotonic profile within the electrolyte. Ion transport in theaqueous electrolyte is the consequence.

A method that is frequently employed for generating such electric fieldsin aqueous systems is application of the decomposition voltage of water.In this case, oxygen is evolved at the anode and hydrogen at thecathode. In the experimental implementation, care must be taken toensure that the gases, and in particular their free radical precursorsdo not come into contact with the molecules to be examined, since thelatter would otherwise be altered chemically. In macroscopic systems,this is done by separating the electrolyte spaces directly before theelectrodes from the electrolyte space between the electrodes, e.g. bymeans of diaphragms. This solution is problematic for microsensors sincediaphragms are not practicable.

One possibility for electrophoresis in microsystems resides inintroducing so-called permeation layers made of hydrophilic polymerbefore the electrodes, in respect of which reference is made to U.S.Pat. No. 5,605,662 A. The mobility of reaction products of theelectrolysis of water and the DNA to be transported is severelyinhibited in this layer, so that an intermixing virtually does not takeplace. The charge transport in the permeation layer is undertaken bysmaller ions.

Although the known method is practicable, the introduction of newpolymer layers makes the production of the microsensor chipsignificantly more complicated and thus more expensive.

SUMMARY

An object of an embodiment of the invention to specify a suitable methodfor transporting the charged molecules via an electric field, in thecase of which no evolution of hydrogen or oxygen occurs at theelectrode. In particular, with utilization of the electrophoresismethod, a corresponding device may be created that manages with standardmaterials and layers of chip production.

In the case of the device according to an embodiment of the invention, aconstruction that is identical, in principle, can be used optionally toperform the method according to at least one embodiment of theinvention. In this case, it is also advantageously possible to combinetwo methods with one another, for example cyclically.

In the application of the electrophoresis method, an embodiment of theinvention makes use of the fact that, in addition to the electrolysis ofwater, other reactions can also be used for generating the electricfield in the analyte solution. An embodiment of the invention proposes ametal/metal ion complex, e.g. copper/copper-histidine complex, as adepolarizer before the electrodes. In the event of positive polarizationof a copper-coated electrode for the purpose of concentrating negativelycharged ions, oxygen is not then evolved; instead, the copper goes intosolution as ion. If a complexing agent for the metal, e.g. histidine forcopper is present there, then the metal iron remains stably in solution.Since e.g. the copper-histidine complex is very stable, theconcentration of the free copper ions remains very small and virtuallyconstant. An influence of the copper ions on the DNA hybridization isthereby avoided.

If the electrode is intended to be negatively polarized in order e.g. toincrease the selectivity of the capture/target molecule binding(stringency treatment), i.e. to remove non-specifically bound,non-complementary sample DNA from the capture DNA, the metal ions arereduced in the presence of a metal ion complex of a sufficiently noblemetal, e.g. copper. Further, they are deposited in the process on theelectrodes (in this case the measuring electrode). Evolution of hydrogenis thereby avoided.

The complexing agent for the metal ion may, under certain circumstances,also serve simultaneously as a buffer. Histidine is used for example asa buffer at pH=7. The copper deposited on the measuring electrodes canbe removed in a washing step by renewed application of negativepotential. A repulsion of the target molecules is prevented by using awashing solution with high ionic strength, so that only e.g. copper inthe form of Cu²⁺ ions is removed, but the target DNA is not moved.

An advantage of an electrophoresis method based on metal/metal ioncomplex resides in the lower voltage required for generating theelectric field. It is lower than the electrolysis voltage of water, sothat the aggressive products of the electrolysis of water cannot arise.A separation of electrolysis space and electrophoresis space thusbecomes unnecessary. The generated field nevertheless suffices totransport the desired molecules in the analyte.

Copper is already used nowadays for interconnects and may be used in thefuture as an electrode material for sensor applications or Microsystemsengineering applications such as micro-electrophoresis. In theproduction of such a microsystem it is therefore possible to haverecourse to cost-effective standard methods of semiconductor technology.

BRIEF DESCRIPTION OF THE DRAWINGS

Further details and advantages of the invention emerge from thefollowing description of figures of example embodiments with referenceto the drawings in conjunction with the patent claims. In the figures:

FIG. 1 shows a basic construction for carrying out the method accordingto an embodiment of the invention,

FIGS. 2 to 4 show cross sections of differently formed arrangements,

FIGS. 5 a and 5 b show, in the case of arrangements in accordance withFIG. 3, in method terms, the enrichment of target molecules from low tohigh concentration,

FIGS. 6 a and 6 b show, in method terms, a situation in accordance withFIG. 5 b, in which, however, non-specific, i.e. non-complementary sampleDNA are also present, which are subjected to a so-called stringencytreatment,

FIG. 7 shows the electrode process in the case of the invention's use ofa sacrificial electrode, and also of a complexing agent,

FIGS. 8 to 10 show plan views of different measuring electrodeconfigurations,

FIG. 11 shows a measuring arrangement with measuring positions arrangednext to one another, in cross section, and

FIG. 12 shows an array arrangement formed from individual positionscorresponding to FIG. 8, in plan view.

The figures will be described together in part.

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

The basic construction of a general arrangement for carrying outbiochemical measurements can be seen from FIG. 1. Element 1 designates aplanar substrate, e.g. made of silicon, on which a thin insulator layer2, e.g. made of silicon oxide (SiO₂), is applied. Two measuringelectrodes 20 and 30, which preferably include noble metal, inparticular gold, are situated on this arrangement. The entire measuringarrangement is in contact with an aqueous solution 15.

The aqueous solution 15 contains negatively charged macromolecules. Thisis illustrated by the bundle structure in FIG. 1, and which aredesignated specifically by 200, 200′ further below in FIG. 5. Thenegatively charged molecules are intended to be transported to themeasuring electrodes 20, 30 and are also referred to hereinafter astarget molecules. In the case of a DNA analysis, the target molecules ofthe DNA to be examined. By way of capture molecules that can beimmobilized e.g. in a hydrogel layer 35, it is possible to attach thetarget DNA for the purpose of measurement in the vicinity of theelectrodes 20, 30.

In the aqueous solution 15 there is furthermore present a material whichis resistant in the aqueous solution and more electronegative than themetal of the measuring electrodes. In the most general case, thematerial is a metal/metal ion (Me/Me⁺) combination, for example Cu/Cu²⁺. Thus, in accordance with the predetermined potential conditions,either metallic copper Cu^(o) is dissolved with two electrons beingreleased or copper(II) ions Cu²⁺ can be deposited with two electronsbeing taken up, in which case the following holds true:Cu^(o)⇄Cu⁺⁺+2e  (2)

In the case of the arrangement in accordance with FIG. 5, in the case ofa copper electrode as sacrificial anode 40, Cu²⁺ can go into solution asa result of a positive potential being applied. As a result, thenegative target molecules 200 are moved there to the copper electrode 40and accumulate in the vicinity thereof and thus also in the region ofthe measuring electrodes 20, 30.

If, with the presence of Cu²⁺ ions in the aqueous solution, a suitablenegative potential is applied to the measuring electrodes 20, 30 inaccordance with FIG. 6, both capture molecule/target DNA bonds breakwhich have a reduced binding strength on account of incompletecomplementarity. At the same time, copper(II) ions (Cu²⁺) are reduced toform metallic copper (Cu^(o)) at the measuring electrodes in theprocess.

The methodical processes in accordance with the alternativesdemonstrated only in principle in FIG. 1 are illustrated with referenceto FIGS. 5 a, 5 b, on the one hand, and 6 a, 6 b on the other hand, andalso FIG. 7. Specifically in FIGS. 5 a to 6 b, a hydrogel layer 35 is ineach case applied above the measuring electrodes 20 and 30, which havesensor surfaces 21 and 31, said hydrogel layer enclosing capturemolecules 100 for target molecules 200 situated outside the hydrogel 35.What is essential in this case is that the capture molecules 100 captureand bind the target molecules 200 and thus supply them for analysis atthe sensor surface 21 and 31, respectively. With regard to thismethodology, reference is made for example to applicant's earlierapplication PCT/DE 02/01982.

The capture molecules 100 may be for example specific thiol-modifiedoligonucleotides. Target molecules 200 that are intended to be bound bythe capture molecules 100 are the DNAs to be analyzed.

In general, a known measuring arrangement exhibits a state in accordancewith FIG. 5 a, in the case of which the target DNA is present only inlow concentration above the capture DNA. It is difficult in this case toattain reliable measurement results. In the case of an arrangement inaccordance with FIG. 5 b, by contrast, the target DNA is present in highconcentration above the capture DNA, this being achieved by way of a DNAenrichment. Good measurement results can be obtained in this state.

In accordance with FIG. 6 a, in addition to the complementary target DNA200, incompletely complementary DNA fragments 200′ also bind to thecapture DNA. By way of a stringency treatment, non-specifically boundDNA can be selectively removed by applying respectively suitablepotentials to the electrodes. The non-specifically bound DNA is thenrepelled on account of its weaker binding forces.

It can be seen from FIG. 1 and also subfigures 5 a and 5 b that adesired enrichment of the target DNA is achieved by applying specificpotentials to the auxiliary electrode 40. In detail, for this purpose anauxiliary electrode 40 made of base metal, for example copper, is chosenand a positive potential is applied to the auxiliary electrode 40. Ifthe entire arrangement is situated in an aqueous solution, Cu²⁺ ions gointo solution. As a result, a field gradient arises and the negativelycharged DNA molecules are attracted.

The latter process is essentially illustrated by FIG. 7. In particular,it can be seen here that the copper ion brought into solution iscomplexed, for which purpose histidine molecules 70 are used.

It can be seen from FIG. 1 and also subfigures 6 a and 6 b that adesired selection of the DNA is achieved by applying specific potentialsto the measuring electrodes 20, 30 and auxiliary electrodes 40, 45. Indetail, the measuring electrodes are polarized negatively and theauxiliary electrodes positively. If the entire arrangement is situatedin an aqueous solution containing copper(II) ions (Cu²⁺), the latter arereduced to metallic copper (Cu^(o)) on the measuring electrodes 20, 30.As a result, a field gradient arises and the negatively charged,incompletely complementary DNA is repelled.

The two alternatives may proceed separately or else in combination.Target molecules are firstly enriched and then selected. However, it isalso possible to perform only a selection.

FIGS. 2 to 4 illustrate different variants of sensor arrangements. InFIG. 2, the measuring electrodes 20, 30 formed from gold have free goldsensor areas 21, 31, to which the capture DNA 100 is bound. As analternative, a hydrogel 35 containing capture DNA 100 is present in FIG.3.

FIG. 4 specifically illustrates an arrangement in which, besides theactual measuring electrodes 20 and 30, a free reaction area 50 made ofgold is furthermore present, to which the capture DNA 100 is bound in adense arrangement. This has the advantage of a high density of captureDNA. However, in the production of the reaction area 50, it is necessaryfirstly to cover the measuring electrodes 20, 30 with copper or the likein order to prevent an attachment of the catcher DNA 100 there. Copperlayers 22 and 32, respectively, are present for this purpose in FIG. 4.

In all of the arrangements in accordance with FIGS. 2 to 4 thesacrificial electrode 40 is in each case arranged in the vicinity of themeasuring electrodes 20 and 30 in order, as a result of copper goinginto solution, to build up the field gradient and thus to effect theenrichment of the target DNA 200 in the vicinity of the measuringelectrodes 20 and 30. The measurement accuracy can thus be considerablyimproved as a result.

FIGS. 8 to 10 illustrate the different variants of measuring sensor inaccordance with FIGS. 2 to 4 in plan view. Specifically in FIG. 8, ameasuring sensor 80 is present which comprises two comb electrodes 82and 83 with intermeshing electrode fingers, a single sacrificialelectrode 84 being arranged annularly around the comb electrodes.

A corresponding arrangement emerges from FIG. 9, here the region of thecomb electrodes being covered with the hydrogel layer 85. A hydrogellayer of this type may be situated over the entire measuringarrangement. Specifically in FIG. 10, reaction areas 86 for theattachment of catcher molecules are additionally present as well.

From the individual sensors in accordance with FIGS. 8 to 10 it ispossible to design arrays having n rows and m columns. FIGS. 11 and 12illustrate a complete arrangement having a multiplicity of measuringsensors 80, 80′, . . . which constitute the n.m array. In this case, itis possible in principle to construct the array with individualpositions corresponding to one of FIGS. 8 to 10, in the case of whicheach individual position has an annular copper sacrificial anode 84. Inthis case the auxiliary electrode 185 is arranged as a further ringaround the entire n.m arrangement with the individual positions.

In accordance with FIG. 11, the complete arrangement 180 is situated ina container, e.g. a through-flow channel 150, with a cover 120, aninflow 121 and an outflow 122.

Example embodiments being thus described, it will be obvious that thesame may be varied in many ways. Such variations are not to be regardedas a departure from the spirit and scope of the present invention, andall such modifications as would be obvious to one skilled in the art areintended to be included within the scope of the following claims.

1. A method for transporting electrically charged molecules in anaqueous solution, the method comprising: arranging, in the vicinity oftwo measuring electrodes, a metallic material which is resistant in theaqueous electrolyte and is more electronegative than that of themeasuring electrode, the metallic material being arranged as anelectrode to which a potential can be applied; and bringing the metallicmaterial, as a result of a positive potential being applied to thearranged electrode, into solution as positive ions, whereby negativelycharged molecules are transported as target molecules in the oppositedirection and are enriched at the measuring electrodes.
 2. The method asclaimed in claim 1, wherein the metal ions going into solution arecomplexed by the presence of a complexing agent, whereby theirconcentration is kept low and virtually constant.
 3. The method asclaimed in claim 2 wherein copper is used as the metallic material, thecopper forming a copper sacrificial anode.
 4. The method as claimed inclaimed in claim 3, wherein—histidine is used as a complexing agent forcomplexing the copper ion.
 5. The method as claimed in claim 1, whereincatcher molecules at an electrode surface are used for detecting thetarget molecules.
 6. The method as claimed in claim 5, whereinthiol-modified capture molecules are used as capture molecules.
 7. Themethod as claimed in claim 5, wherein hydrogel-bound molecules are usedas capture molecules.
 8. The method as claimed in claim 1, wherein anelectrophoresis method is performed.
 9. The method as claimed in claim1, wherein a DNA analysis of DNA fragments is effected.
 10. The methodas claimed in claim 9, wherein the enriched molecules are detected astarget molecules during the DNA analysis.
 11. The method as claimed inclaim 8 wherein the selectivity of the process is increased bypolarization of the electrodes used for the electrophoresis or DNAanalysis.
 12. A method for binding-specific separation of electricallycharged molecules in an aqueous solution, during the operation of asensor with a cycling process between two measuring electrodes, themethod comprising: situating metal ions in the aqueous solution;depositing, as a result of a negative potential being applied to themeasuring electrodes, the metal ion as metal at the measuringelectrodes, whereby negatively charged molecules bound in the vicinityof the measuring electrodes are transported away from the measuringelectrodes as target molecules with a sufficiently low binding energy.13. The method as claimed in claim 12, wherein copper is used as metalions and gold is used as measuring electrodes.
 14. The method as claimedin claim 12, wherein the molecules transported away from the measuringelectrodes are those target molecules which are not intended to bedetected during a DNA analysis.
 15. A device for carrying out the methodas claimed in claim 1 having an arrangement comprising the measuringelectrodes for electrochemical measurement in an aqueous solution, therebeing present in the aqueous solution at least one of metal ions andaccumulations of metal made of more electronegative material than thatof the measuring electrodes, the material being resistant in aqueoussolution.
 16. The device as claimed in claim 15, wherein the measuringelectrodes comprise noble metal.
 17. The device as claimed in claim 15,wherein the metal is copper and forms a sacrificial electrode.
 18. Thedevice as claimed in claim 16, wherein the measuring electrodes made ofgold have a sensor surface to which capture molecules for the target DNAare bound.
 19. The device as claimed in claimed 15, wherein themeasuring electrodes form an interdigital structure including combelectrodes with intermeshing electrode fingers.
 20. The device asclaimed in claim 17, wherein the sacrificial electrode is arrangedannularly around the comb electrodes.
 21. The device as claimed in claim15, wherein a hydrogel layer for binding the capture molecules isarranged on the measuring electrodes.
 22. The device as claimed in claim15, wherein the measuring electrodes are assigned separate reactionareas for attachment of the capture molecules.
 23. The device as claimedin claim 19, wherein an array having m rows and n columns is formed byindividual interdigital structures with sacrificial electrode.
 24. Thedevice as claimed in claim 23, wherein an auxiliary electrode withrespect to the individual sacrificial electrodes runs annularly aroundthe m.n array.
 25. The method as claimed in claim 1, wherein the methodis for transporting electrically charged molecules in an aqueoussolution during the operation of a DNA sensor with a redox cyclingprocess between the two measuring electrodes.
 26. The method as claimedin claim 1, wherein copper is used as the metallic material, the copperforming a copper sacrificial anode.
 27. A device for carrying out themethod as claimed in claim 12, having an arrangement comprising themeasuring electrodes for electrochemical measurement in an aqueoussolution, there being present in the aqueous solution at least one ofmetal ions and accumulations of metal made of more electronegativematerial than that of the measuring electrodes, the material beingresistant in aqueous solution.
 28. The device as claimed in claim 16,wherein the measuring electrodes form an interdigital structureincluding comb electrodes with intermeshing electrode fingers.
 29. Thedevice as claimed in claim 18, wherein the measuring electrodes form aninterdigital structure including comb electrodes with intermeshingelectrode fingers.